MicroRNAs (miRNAs) are non-protein coding RNAs, generally of between about 19 to about 25 nucleotides (commonly about 20-24 nucleotides in plants), that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways (Bartel (2004) Cell, 116:281-297). In some cases, miRNAs serve to guide in-phase processing of siRNA primary transcripts (see Allen et al. (2005) Cell, 121:207-221).
Many microRNA genes (MIR genes) have been identified and made publicly available in a database (‘miRBase”, available on line at microrna.sanger.ac.uk/sequences; also see Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MicroRNAs were first reported from nematodes and have since been identified in other invertebrates; see, for example, Lee and Ambros (2001) Science, 294:862-864; Lim et al. (2003) Genes Dev., 17:991-1008; Stark et al. (2007) Genome Res., 17:1865-1879. MIR genes have been reported to occur in intergenic regions, both isolated and in clusters in the genome, but can also be located entirely or partially within introns of other genes (both protein-coding and non-protein-coding). For a recent review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385. Transcription of MIR genes can be, at least in some cases, under promotional control of a MIR gene's own promoter. The primary transcript, termed a “pri-miRNA”, can be quite large (several kilobases) and can be polycistronic, containing one or more pre-miRNAs (fold-back structures containing a stem-loop arrangement that is processed to the mature miRNA) as well as the usual 5′ “cap” and polyadenylated tail of an mRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385.
Maturation of a mature miRNA from its corresponding precursors (pri-miRNAs and pre-miRNAs) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are believed to be largely processed to the mature miRNA entirely in the nucleus, whereas in animal cells, the pri-miRNA transcript is processed in the nucleus by the animal-specific enzyme Drosha, followed by export of the pre-miRNA to the cytoplasm where it is further processed to the mature miRNA. Mature miRNAs in plants are typically 21 nucleotides in length, whereas in animals 22 nucleotide long miRNAs are most commonly found. For a recent review of miRNA biogenesis in both plants and animals, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385. Additional reviews on miRNA biogenesis and function are found, for example, in Bartel (2004) Cell, 116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol., 16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol., 7:512-520. Furthermore, although one recent report describes a miRNA (miR854) from Arabidopsis that also is found in animals (Arteaga-Vazquez et al. (2006) Plant Cell, 18:3355-3369), miRNA conservation generally appears to be kingdom-specific. Animal miRNAs have many characteristic dissimilar to their plant counterparts, including shorter miRNA precursor fold-backs (about 90 nucleotides in animals versus about 180 nucleotides in plants) with the mature miRNA sequence tending to be found at the base of the stem, a higher number of mismatches within the foldback, and derivation from from polycistronic messages. Whereas animal miRNAs generally anneal imperfectly to the 3′ untranslated region (UTR) of their target mRNA, most plant miRNAs are characterized by having perfect or near-perfect complementarity to their target sequence, which is usually in the coding region, with only a few examples of miRNAs having binding sites within the UTRs of the target mRNA; see Rhoades et al. (2002) Cell, 110:513-520; Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53. These significant differences between plant and animal miRNAs make it generally unlikely that miRNAs will be processed and function across kingdoms.
Transgenic expression of miRNAs (whether a naturally occurring sequence or an artificial sequence) can be employed to regulate expression of the miRNA's target gene or genes. Inclusion of a miRNA recognition site in a transgenically expressed transcript is also useful in regulating expression of the transcript; see, for example, Parizotto et al. (2004) Genes Dev., 18:2237-2242. Recognition sites of miRNAs have been validated in all regions of an mRNA, including the 5′ untranslated region, coding region, and 3′ untranslated region, indicating that the position of the miRNA target site relative to the coding sequence may not necessarily affect suppression (see, e. g., Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290, Sunkar and Zhu (2004) Plant Cell, 16:2001-2019). Because miRNAs are important regulatory elements in eukaryotes, transgenic suppression of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of MIR genes can have very specific expression patterns (e. g., cell-specific, tissue-specific, temporally specific, or inducible), and thus are useful in recombinant constructs to induce such specific transcription of a DNA sequence to which they are operably linked. Various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication 2006/0200878 A1, incorporated by reference herein. Non-limiting examples of these utilities include: (1) the expression of a native miRNA or miRNA precursor sequence to suppress a target gene; (2) the expression of an engineered (non-native) miRNA or miRNA precursor sequence to suppress a target gene; (3) expression of a transgene with a miRNA recognition site, wherein the transgene is suppressed when the mature miRNA is expressed; (4) expression of a transgene driven by a miRNA promoter.
Animal miRNAs have been utilized as precursors to express specific miRNAs in animal cells; for example, the human miR-30 precursor was expressed as the native sequence and as a modified (artificial or engineered) miRNA in cultured cells (Zeng et al. (2002) Mol. Cell, 9:1327-1333, and Zeng et al. (2005) J. Biol. Chem., 280:27595-27603). A single mature miRNA is precisely processed from a given precursor, and therefore such “artificial” or engineered miRNAs offer an advantage over double-stranded RNA (dsRNA) in that only a specific miRNA sequence is expressed, limiting potential off-target effects. Although animal miRNAs typically interact with imperfect target sequences in the 3′ UTR, synthetic miRNAs with perfect target complementarity also can guide target cleavage (see Zeng et al. (2003) RNA, 9:112-123 and Zeng et al. (2003) Proc. Natl. Acad. Sci. U.S.A, 100:9779-9784).
Small RNAs, referred to as short interfering RNAs (siRNAs) and micro RNAs (miRNAs), have been shown to regulate gene expression in plants and animals (Valencia-Sanchez et al. (2006) Genes Dev., 20:515-524; Nelson et al. (2003) Trends Biochem. Sci., 28:534-540). Experimental alteration of siRNA levels result in phenotypic effects in nematodes (Timmons and Fire (1998) Nature, 395:8543). A plant that transgenically expressed siRNA complementary to the root-knot nematode 16D10 gene was shown to have resistance to four species of root-knot nematodes (Huang et al. (2006) Proc. Natl. Acad. Sci. U.S.A, 103:14302-14306). This invention discloses the use of recombinant invertebrate miRNAs expressed in planta to similarly regulate expression in an invertebrate that ingests the miRNAs.
This invention discloses recombinant DNA constructs encoding invertebrate mature miRNAs and their miRNA precursors, which are designed to be expressed in planta. In some embodiments, the invertebrate miRNA precursors are engineered to express artificial miRNAs designed to suppress or silence specific invertebrate genes and thereby confer upon a plant expressing the miRNAs resistance to an invertebrate that ingests the miRNAs. In many cases, RNAi (siRNA or miRNA) transcripts that are intended to suppress an invertebrate target are preferably ingested by the invertebrate as larger transcripts, that is, larger than the 21 to 24 nucleotide fragments typically resulting from in planta processing. Thus, RNAi transcripts intended for ingestion are preferably designed to be resistant to in planta processing. The recombinant invertebrate miRNAs of this invention are preferably resistant to the plant-specific endogenous miRNA processing (in comparison to plant-derived miRNAs), but are preferably readily recognized in invertebrate cells where they are processed to the mature miRNA.